A method for purifying crude sodium mannose phosphate

Abstract

This invention belongs to the field of biotechnology separation and discloses a purification method for crude sodium mannose phosphate. The method includes: centrifuging and ultrafiltration of the sodium mannose phosphate reaction solution obtained from a catalytic reaction to obtain a membrane-filtered supernatant; purifying the supernatant using a two-step electrodialysis method, repeating the purification 2-5 times to obtain an aqueous solution of sodium mannose phosphate; and then drying the solution to obtain a high-purity sodium mannose phosphate product. Different ion exchange membranes are used in the two-step electrodialysis method. This method, through electrodialysis, combines a homogeneous membrane and an alloy membrane to achieve effective separation of nonionic impurities such as mannose, as well as various phosphates, from sodium mannose phosphate.

Description

Technical Field

[0001] This invention belongs to the field of biological separation and purification technology, specifically relating to a purification method for crude sodium mannose phosphate. Background Technology

[0002] Sodium mannose phosphate is generally produced through chemical synthesis or enzymatic catalysis. During the production process, the product system contains some unreacted hexoses, as well as large amounts of orthophosphates, metaphosphates, and polyphosphates. The presence of hexoses not only reduces product purity but also easily causes discoloration during application, such as in the heating processes of cosmetic formulations, leading to changes in product color. Phosphates, due to their unique physicochemical properties, are the most significant destabilizing factor in the product, causing turbidity during concentration, precipitation at low temperatures, and sedimentation during long-term storage, greatly affecting production and application.

[0003] Currently, the main desalination methods used are precipitation and ion exchange. However, because mannose phosphate itself also has phosphate groups, precipitation has poor selectivity, leading to partial precipitation of mannose phosphate and significant product loss. Ion exchange is limited by the capacity of the ion exchange resin, requiring sample dilution and subsequent concentration after desalination, making the entire process complex. Ion exchange resins also require high selectivity for mannose phosphate and phosphates, and the large amounts of eluent and waste resin increase environmental pressure. Existing methods for separating mannose primarily involve ion exchange, which uses anion exchange resin to adsorb mannose phosphate followed by elution to separate and remove mannose not adsorbed by the resin. The advantages and disadvantages of this method are the same as those of ion exchange in desalination processes.

[0004] Based on the above, currently available commercially available sodium mannose phosphate products are either mixed aqueous solutions of sodium mannose phosphate and mannose, with limited applications, or pure sodium mannose phosphate, which is expensive and generally used as a testing standard. There are currently no relatively inexpensive sodium mannose phosphate products with high purity that can be widely used in food, health products, pharmaceuticals, and cosmetics. Summary of the Invention

[0005] (I) Purpose of the Invention

[0006] The purpose of this invention is to provide a purification method for crude sodium mannose phosphate. This method uses electrodialysis, combining a homogeneous membrane and an alloy membrane, to effectively separate nonionic impurities such as mannose, various phosphates, and other small molecular weight ions from sodium mannose phosphate.

[0007] (II) Technical Solution

[0008] To address the above problems, a first aspect of the present invention provides a method for purifying crude sodium mannose phosphate, comprising:

[0009] The sodium mannose phosphate reaction solution obtained from the catalytic reaction was centrifuged and ultrafiltered to obtain a clear solution after membrane filtration.

[0010] The membrane-passed supernatant was purified by a two-step electrodialysis method. After 2 to 5 consecutive purifications, an aqueous solution of sodium mannose phosphate was obtained. The solution was then dried to obtain a high-purity sodium mannose phosphate product. Different ion exchange membranes were used in the two-step electrodialysis method.

[0011] Furthermore, in the two-step electrodialysis method, the first step of electrodialysis uses an alloy membrane, and the second step of electrodialysis uses a homogeneous membrane.

[0012] Furthermore, the alloy film uses a polymer alloy as the substrate and has a molecular weight of 400 Da to 450 Da.

[0013] Furthermore, the homogeneous membrane uses styrene-divinylbenzene as the substrate and transmits molecules with a molecular weight of 250 Da to 300 Da.

[0014] Furthermore, the alloy mold and the homogeneous membrane are cation exchange membranes or anion exchange membranes.

[0015] Furthermore, in the first step of electrodialysis, the membrane-passed supernatant is used as the first dilute solution and deionized water is used as the first concentrated solution.

[0016] Furthermore, in the second step of electrodialysis, the first concentrated solution is used as the second dilute solution, and deionized water is used as the second concentrated solution.

[0017] Furthermore, the first step of electrodialysis takes 4 to 9 hours, and the second step of electrodialysis takes 0.5 to 3 hours.

[0018] Furthermore, in both steps of the electrodialysis method, PBS is used as the protective solution, with a concentration of 0.3 mol / L and a pH of 7.0.

[0019] Furthermore, the concentration of the sodium mannose phosphate reaction solution is 50–100 g / L.

[0020] (III) Beneficial Effects

[0021] The above-mentioned technical solution of the present invention has the following beneficial technical effects: The present invention provides a purification method for crude sodium mannose phosphate, which adopts a two-step electrodialysis method. By using different ion exchange membranes in combination, non-ionic impurities such as mannose, phosphate, hypophosphite, and other small molecular weight ions are separated from sodium mannose phosphate. The principle is as follows: First, the sodium mannose phosphate reaction solution obtained by the catalytic reaction is centrifuged and ultrafiltered to obtain a membrane-passed clear liquid; then, the membrane-passed clear liquid is purified by a two-step electrodialysis method. Taking advantage of the difference that the molecular weight of mannose phosphate ions is 282 Da, while the molecular weight of phosphate ions is less than 200 Da, and that mannose is a non-ionic molecule, non-ionic substances such as mannose are first separated using an alloy membrane, and then phosphate is separated using a homogeneous membrane. Through the above two processes, the separation of mannose phosphate, phosphate, and mannose can be achieved. This method is applicable to the removal of phosphates, hypophosphites, other small molecular weight ions, and nonionic impurities such as mannose from the membrane-filtered supernatant obtained after centrifugation and ultrafiltration of the reaction solution during the preparation of sodium mannose phosphate. Furthermore, this method is simple to operate, highly efficient in purification, environmentally friendly, and low in cost. After 2-5 consecutive purification cycles, an aqueous solution of sodium mannose phosphate is obtained, which is then dried to obtain a high-purity sodium mannose phosphate product with a purity of up to 99.9%, effectively meeting the demand for high-purity sodium mannose phosphate in research, production, and applications. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that these descriptions are merely exemplary and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concepts of the invention.

[0023] I. The materials, instruments, and methods used in this invention include:

[0024] Electrodialysis unit: EX-3BT or EX-4S;

[0025] Electrodialysis membranes: styrene-divinylphenyl cation and anion membranes (homogeneous membranes); polymer alloy cation and anion membranes (alloy membranes);

[0026] Water: All water used in the embodiments shall meet the Class I water requirements of GB / T 6682. Unless otherwise specified, the term "water" as used in this invention refers to ultrapure water.

[0027] In this invention, the terms "sodium mannose phosphate" and "mannose phosphate", "sodium mannose-6-phosphate", "sodium mannose-6-phosphate", "mannose-6-phosphate", "sodium mannose phosphate", "mannose-6-phosphate", and "M6P" have the same meaning and can be used interchangeably.

[0028] The detection methods for each substance in this invention include:

[0029] Detection of sodium mannose phosphate content: Use the K-MANGL kit for detecting mannose content, but without adding the reagent ATP, so that it can only detect the content of mannose phosphate. Perform the remaining operations according to the kit instructions.

[0030] Detection of mannose content: Using the K-MANGL kit for detecting mannose content, the procedure was performed according to the kit instructions to obtain the mannose content. Then, based on the result of the sodium mannose phosphate content detection, the actual mannose content in the original sample was obtained by subtracting the sodium mannose phosphate content detection value from the mannose content detection value.

[0031] Detection of phosphate content: The molybdenum blue method was used.

[0032] The present invention provides a method for purifying crude sodium mannose phosphate, specifically comprising:

[0033] S1, the sodium mannose phosphate reaction solution obtained from the catalytic reaction is centrifuged and ultrafiltered to obtain a membrane-filtered clear solution; the concentration of the sodium mannose phosphate reaction solution is 50-100 g / L.

[0034] S2, the clarified solution is purified using a two-step electrodialysis method, and after 2-5 consecutive purifications, an aqueous solution of sodium mannose phosphate is obtained. This solution is then dried to obtain a high-purity sodium mannose phosphate product. Different ion exchange membranes are used in the two-step electrodialysis method. The first step of the electrodialysis uses an alloy membrane, which is based on a high-molecular-weight alloy and has a permeable molecular weight of 400-450 Da. The alloy membrane is used in the first step to remove mannose, trace amounts of other non-ionic substances, and large molecular weight ions. Taking advantage of the difference that mannose phosphate ions have a molecular weight of 282 Da, while phosphate ions have a molecular weight of less than 200 Da, and that mannose is a non-ionic molecule; the alloy membrane can retain molecular weights of 400 Da to 450 Da, allowing mannose phosphate ions and phosphate ions to pass through, but not mannose that cannot migrate under the influence of current, or other residual large molecular ions. Therefore, this first step uses the alloy membrane to separate non-ionic substances such as mannose. The first step of electrodialysis takes 4 to 9 hours to ensure that mannose phosphate ions completely permeate through the alloy membrane. A homogeneous membrane is used in the second step of electrodialysis. The homogeneous membrane uses styrene-divinylbenzene as the substrate and allows the permeation of molecular weights of 250 Da to 300 Da. In the first step of electrodialysis, the supernatant used as the first dilute chamber solution, deionized water as the first concentrated chamber solution, and PBS as the protective solution. The second step uses a homogeneous membrane because it can retain molecular weights of 250 Da to 300 Da. It allows phosphate ions to pass through but not mannose phosphate ions (when the homogeneous membrane allows a molecular weight of around 300 Da, the molecular weight of mannose phosphate ions is 280 Da, not significantly different from 300 Da; although mannose phosphate ions can pass through the homogeneous membrane, the percentage passing through will be relatively small). The electrodialysis time in the second step is 0.5 to 3 hours to ensure the permeation loss rate of mannose phosphate ions within this short time period. The alloy mold and the homogeneous membrane are either cation exchange membranes or anion exchange membranes. In the second electrodialysis step, the first concentrated solution is used as the second dilute solution, deionized water is used as the second concentrated solution, and PBS is used as the protective solution. In both electrodialysis steps, PBS is used as the protective solution, with a concentration of 0.3 mol / L and a pH of 7.0.

[0035] Alternatively, in the two-step electrodialysis of this invention, a homogeneous membrane can be used in the first step and an alloy membrane in the second step without affecting the experimental results. However, in actual operation, using an alloy membrane to remove mannose in the first step reduces the discoloration of the material due to heating. Therefore, using an alloy membrane in the first step and a homogeneous membrane in the second step is the preferred method of this invention.

[0036] II. Implementation Methods

[0037] Example 1

[0038] In the production process of sodium mannose phosphate, the reaction solution after the enzyme-catalyzed reaction is centrifuged and ultrafiltered, and an appropriate amount of the membrane-passed supernatant is collected. The content of sodium mannose phosphate is analyzed to be 78.60 g / L, mannose content is 23.77 g / L, and phosphate ion content is 25.44 g / L. 1000 mL of this supernatant is added to the dilute chamber of the electrodialysis machine as the dilute solution, and the alloy membrane is used as the electrodialysis membrane for the first electrodialysis. Separately, 500 mL of deionized water is added to the concentrate chamber as the concentrate solution, and 1000 mL of 0.3M PBS is added to the electrode water chamber as the protective solution. Electrodialysis parameters: Electrodialysis machine model, EX-3BT; total effective membrane area 0.055 m². 2 The voltage was 15V, the current threshold was set to 2.2A, the material flow rate was 3L / min, and the electrodialysis time was 6h. The first electrodialysis used an alloy membrane (alloy membrane electrodialysis), and the second electrodialysis used a homogeneous membrane (homogeneous membrane electrodialysis). The concentrate from the first electrodialysis was added to the dilute solution tank as the dilute solution for the second electrodialysis. The homogeneous membrane was used as the electrodialysis membrane for the second electrodialysis. Separately, 500mL of deionized water was added to the concentrate tank as the concentrate, and 1000mL of 0.3M PBS was added to the electrode water tank as a protective solution. The parameters for the second electrodialysis were the same as for the first electrodialysis, and the electrodialysis time was 1h. The content of each component in the dilute and concentrate solutions from both electrodialysis processes was measured, and the results are shown in Table 1. The dilute solution from the second electrodialysis was the purified product, with a volume of 559 mL. The purity of sodium mannose phosphate was 87.79%, and the overall yield of the two electrodialysis cycles, based on sodium mannose phosphate, was 76.50%.

[0039] The purity of sodium mannose phosphate is calculated based on its content in the dilute chamber solution after homogeneous membrane electrodialysis, using the following formula:

[0040] The purity of sodium mannose phosphate = sodium mannose phosphate content / (sodium mannose phosphate content + mannose content + phosphate content) × 100%;

[0041] Total electrodialysis yield = (sodium mannose phosphate content in the dilute solution after homogeneous membrane electrodialysis × volume of dilute solution after homogeneous membrane electrodialysis) ÷ (sodium mannose phosphate content in the dilute solution before alloy membrane electrodialysis × volume of dilute solution before alloy membrane electrodialysis) × 100%.

[0042] Table 1. Content determination results before and after electrodialysis in Example 1

[0043]

[0044]

[0045] Example 2

[0046] Take 1000 mL of the same batch of membrane-exposed solution as in Example 1, and operate under the electrodialysis conditions of Example 1. The first electrodialysis time is 9 h, and the second electrodialysis time is 0.5 h. The content of each component in the dilute and concentrated solutions of the two electrodialysis processes is determined, and the results are shown in Table 2. The dilute solution of the second electrodialysis is the purified product, with a volume of 579 mL and a sodium mannose phosphate purity of 82.00%. The overall yield of the two electrodialysis processes, calculated based on sodium mannose phosphate, is 86.45%.

[0047] Table 2. Content determination results before and after electrodialysis in Example 2.

[0048]

[0049] Example 3

[0050] Take 1000 mL of the same batch of membrane-passed supernatant as in Example 1, and operate under the electrodialysis conditions of Example 1. The first electrodialysis time is 4 h, and the second electrodialysis time is 3 h. The content of each component in the dilute and concentrate solutions of the two electrodialysis processes is measured, and the results are shown in Table 3. The dilute solution of the second electrodialysis is the purified product, with a volume of 513 mL and a sodium mannose phosphate purity of 93.51%. The overall yield of the two electrodialysis processes, calculated based on sodium mannose phosphate, is 59.25%.

[0051] Table 3. Content determination results before and after electrodialysis in Example 3.

[0052]

[0053] Example 4

[0054] Eight sample solutions from different batches than those in Example 1 were taken, and 1000 mL of the supernatant was collected from each. The sodium mannose phosphate content was determined to be 78.60 g / L, the mannose content 23.77 g / L, and the phosphate ion content 25.44 g / L. The first purification was performed according to the electrodialysis conditions and time described in Example 1. The content of each component in the dilute solution after the second electrodialysis was determined, and the purity and yield were calculated. The results are shown in Table 4.

[0055] Table 4. Results of each group after the first purification.

[0056]

[0057]

[0058] The dilute solution from the second electrodialysis after the first purification was mixed thoroughly to obtain 4436 mL of a mixed solution containing 115.01 g / L sodium mannose phosphate, 6.23 g / L mannose, and 8.22 g / L phosphate ions. The purity and yield demonstrate that the method described in this invention has good reproducibility. The average purity of the eight experiments was 88.71%, and the average yield was 76.53%.

[0059] Four separate 1000 mL portions of the above mixed solution were taken and purified a second time according to the electrodialysis conditions and time specified in Example 1. The content of each component in the dilute solution after the second electrodialysis was determined, and the purity and yield were calculated. The results are shown in Table 5.

[0060] Table 5. Results of each group after the second purification.

[0061]

[0062] The dilute solution from the second electrodialysis after the second purification was mixed thoroughly to obtain a mixed solution of 2457 mL containing sodium mannose phosphate (147.94 g / L), mannose (1.08 g / L), and phosphate (2.37 g / L). The average purity of the four experiments was 97.72%, and the average yield was 79.02%. Two 1000 mL portions of the above mixed solution were taken and purified a third time according to the electrodialysis conditions and time described in Example 1. The content of each component in the dilute solution after the second electrodialysis was measured, and the purity and yield were calculated. The results are shown in Table 6.

[0063] Table 6. Results of each group after the third purification.

[0064]

[0065] The dilute solution from the second electrodialysis after the third purification was mixed thoroughly to obtain 1307 mL of a mixed solution containing 194.67 g / L sodium mannose phosphate, 0.05 g / L mannose, and 0.34 g / L phosphate ions. The average purity of the two experiments was 99.80%, and the average yield was 86.00%. 1000 mL of the above mixed solution was taken and purified a fourth time according to the electrodialysis conditions and time described in Example 1. The content of each component in the dilute solution after the second electrodialysis was measured, and the purity and yield were calculated. The results are shown in Table 7.

[0066] Table 7. Results of each group after the fourth purification.

[0067]

[0068] After four purification processes, a product solution with a purity of 99.87% was obtained, with an overall yield of 89.28%.

[0069] This invention provides a purification method for crude sodium mannose phosphate, employing a two-step electrodialysis method that combines an alloy membrane and a homogeneous membrane to simultaneously complete the desalting and desaccharification processes. During purification, the alloy membrane first separates nonionic substances such as mannose, and then the homogeneous membrane separates the phosphate. Through these two processes, the separation of mannose phosphate, phosphate, and mannose is achieved. The principle is based on the difference that mannose phosphate ions have a molecular weight of 282 Da, while phosphate ions have a molecular weight less than 200 Da, and that mannose is a nonionic molecule. The alloy membrane has a molecular weight cutoff of 400 Da to 450 Da, allowing mannose phosphate ions and phosphate ions to pass through, but not mannose that cannot migrate under the influence of current, or other residual large molecular ions. The homogeneous membrane has a molecular weight cutoff of 250 Da to 300 Da, allowing phosphate ions to pass through, but not mannose phosphate ions. After 2-5 consecutive purifications, an aqueous solution of sodium mannose phosphate is obtained, which is then dried to obtain a high-purity sodium mannose phosphate product. Using the electrodialysis method of this invention, a single purification process can achieve a purity of 83-85%, a second purification process can achieve a purity of over 98%, and a five-stage purification process can achieve a purity of 99.9%. The purification method of this invention is simple to operate, highly efficient, environmentally friendly, and low in cost. It is suitable for the effective removal of nonionic impurities such as mannose, phosphates, hypophosphites, and other small molecular weight ions from the product during the preparation and production of sodium mannose phosphate.

[0070] It should be understood that the specific embodiments described above are merely illustrative or explanatory of the principles of the invention and do not constitute a limitation thereof. Therefore, any modifications, equivalent substitutions, improvements, etc., made without departing from the spirit and scope of the invention should be included within the protection scope of the invention. Furthermore, the appended claims are intended to cover all variations and modifications falling within the scope and boundaries of the appended claims, or equivalent forms of such scope and boundaries. Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the implementation or testing of the invention, preferred methods and materials are now described. The invention has been described above with reference to embodiments thereof. However, these embodiments are merely illustrative and not intended to limit the scope of the invention. The scope of the invention is defined by the appended claims and their equivalents. Various substitutions and modifications can be made by those skilled in the art without departing from the scope of the invention, and all such substitutions and modifications should fall within the scope of the invention. Although embodiments of the invention have been described in detail, it should be understood that various changes, substitutions, and alterations can be made to the embodiments of the invention without departing from the spirit and scope thereof. Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for purifying crude sodium mannose phosphate, characterized in that, include: The sodium mannose phosphate reaction solution obtained from the catalytic reaction was centrifuged and ultrafiltered to obtain a clear solution after membrane filtration. The membrane-passed supernatant was purified by a two-step electrodialysis method. After 2 to 5 consecutive purifications, an aqueous solution of sodium mannose phosphate was obtained. The solution was then dried to obtain a high-purity sodium mannose phosphate product. Different ion exchange membranes were used in the two-step electrodialysis method.

2. The purification method for crude sodium mannose phosphate according to claim 1, characterized in that, In the two-step electrodialysis method, the first step of electrodialysis uses an alloy membrane, and the second step of electrodialysis uses a homogeneous membrane.

3. The purification method for crude sodium mannose phosphate according to claim 2, characterized in that, The alloy film uses a polymer alloy as the substrate and has a molecular weight of 400 Da to 450 Da.

4. The purification method for crude sodium mannose phosphate according to claim 2, characterized in that, The homogeneous membrane uses styrene-divinylbenzene as the substrate and transmits molecules with molecular weights of 250 Da to 300 Da.

5. The purification method for crude sodium mannose phosphate according to claim 2, characterized in that, The alloy mold and the homogeneous membrane are either cation exchange membranes or anion exchange membranes.

6. The purification method for crude sodium mannose phosphate according to claim 2, characterized in that, In the first step of electrodialysis, the membrane-passed supernatant is used as the first dilute solution and deionized water is used as the first concentrated solution.

7. The purification method for crude sodium mannose phosphate according to claim 6, characterized in that, In the second step of electrodialysis, the first concentrated solution is used as the second dilute solution, and deionized water is used as the second concentrated solution.

8. The purification method for crude sodium mannose phosphate according to claim 2, characterized in that, The first step of electrodialysis takes 4 to 9 hours, and the second step of electrodialysis takes 0.5 to 3 hours.

9. The purification method for crude sodium mannose phosphate according to claim 1, characterized in that, In both steps of the electrodialysis method, PBS is used as the protective solution, with a concentration of 0.3 mol / L and a pH of 7.

0.

10. The purification method for crude sodium mannose phosphate according to claim 1, characterized in that, The concentration of the sodium mannose phosphate reaction solution is 50–100 g / L.

Images